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Amorphous no more: subdiffraction view of the pericentriolar material architecture Vito Mennella1, David A. Agard1, Bo Huang2,3, and Laurence Pelletier4,5 1

Department of Biochemistry and Biophysics, HHMI and University of California, San Francisco, San Francisco, CA, USA Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA 4 Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada 5 Department of Molecular Genetics, University of Toronto, Toronto, Canada 2

The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The pericentriolar material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules and acts as a nexus for molecular signaling. Although great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance on electron microscopy (EM) have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supramolecular organization of organelles in cells. Centrosome structure and function The microtubule cytoskeleton is a dynamic network of filaments comprising a/b-tubulin polymers that mediates the transport of organelles, protein complexes and segregation of genetic material through the mitotic spindle apparatus in eukaryotes. In most animal cells, the microtubule network is organized by the centrosome, a nonmembrane-bound organelle comprising two molecular assemblies with distinct but integrated functions: the centriole, a nine-fold symmetric cylindrical structure encircled by microtubule blades; and the PCM, traditionally described as an amorphous, electron-dense material that surrounds the centrioles [1,2]. Most cells have two centrosomes, each containing a mother centriole and, following duplication, a daughter, whose dimensions can reach approximately 200 nm in diameter and typically between 200 and 400 nm in length depending on species, Corresponding author: Mennella, V. ([email protected]). Keywords: centrosomes; cilia; pericentriolar material; super-resolution microscopy; mitosis; cell cycle. 0962-8924/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.10.001

cell type, and the cell cycle phase. In cells with specialized functions, the size and number of centrioles can vary, reaching hundreds in number and microns in length [3]. The two centrosomes, which differ in age as the centrioles, can be structurally distinguished by the presence of distal and subdistal appendages, which confer to the older of the mother centrioles specialized microtubule-anchoring capabilities [1,2,4,5]. In specialized non-dividing cells, the mother centriole docks at the cell membrane and undergoes extensive structural modification to form the basal body [6], which provides a template for the formation of the microtubule-based axoneme, a structure critical for signal transduction via cilia and for cellular movement as flagella [7]. The two structural elements of centrosomes, the centrioles and the PCM, have defined and interconnected functional roles. The centrioles act as a structural scaffold to promote the organization of the PCM [8–10]. Conversely, the primary functional role of the PCM is to anchor microtubules directly or through microtubule-nucleating centers (g-tubulin ring complexes [gTuRCs] [11]). At the onset of mitosis, in a process termed centrosome maturation, the PCM increases in size and microtubule-nucleation capacity through an increase in the recruitment of gTuRCs from the cytosol. This leads to an increase in the nucleation of both astral and spindle microtubules, which drive spindle formation, orientation, and, subsequently, cytokinesis [1]. The PCM also plays a role in the duplication of centrioles [12,13] and probably in cilia formation and disassembly [14,15], further underscoring the interplay between the two assemblies. In addition to its more specific roles, the PCM broadly functions as a signaling and docking station to regulate and redistribute protein assemblies through microtubule transport by motor proteins or through association with microtubule plus ends [16,17]. PCM proteins have been implicated in cell cycle progression, protein degradation, DNA damage, and hedgehog signaling [18– 23], but it remains unclear to what extent the PCM scaffold functions directly in the assembly and regulation of such complexes. Interestingly, the centrosome can be essential, critical, or dispensable for accurate division depending on the Trends in Cell Biology xx (2013) 1–10

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Review specific cell and tissue and their reliance on oriented cell divisions. Thus eliminating the centrosome has severe but distinct consequences for tissue organization, homeostasis, and development in different species. Mice with mutations in many centrosomal proteins have dramatic developmental defects, such as microcephaly. Conversely, flies and worms do not proceed beyond early embryogenesis without centrosomes and die after a few cellular divisions [10,24– 26]. Surprisingly, when flies survive the early embryonic stages with a parental contribution of centrosomes, they are able to develop into adults and eventually die only after eclosion due to lack of coordination [10]. Numerical and structural centrosome aberrations are hallmarks of cells from solid tumors and are thought to contribute to genetic instability and cancer initiation and progression [2,26,27]. Moreover, several hereditary diseases – collectively named ciliopathies – have been mapped to mutations in human genes whose products encode centrosomal proteins with a role in cilia formation. This highlights the necessity to further comprehend how defects in centrosome organization and duplication are linked to disease states [5,26,28]. In recent years, mass spectrometry analyses of isolated centrosomes from different cells and organisms have defined the centrosome proteome [29,30]. A critical task in centrosome biology is to integrate this information into defined protein molecular complexes and understand how their structure influences centrosomes activity in cells [31]. Toward this end, studies by EM have provided insights into the ultrastructural organization of the centriole cartwheel [32,33] and procentrioles [34,35] or gTuRCs [11]. However, even with the use of electron tomography methods, which early on revealed ring-shaped proteinaceous structures embedded within the PCM [36], the molecular architecture and higher-order spatial organization of the PCM has remained, until recently, murky. Here we describe the current understanding of centrosome architecture and the molecular mechanisms that govern PCM assembly. We emphasize recent efforts to examine quantitatively the structure of the PCM using subdiffraction-resolution microscopy imaging methods – 3D structured-illumination microscopy (3DSIM), stochastic optical-reconstruction microscopy (STORM) – and 3D subvolume alignment and averaging. Recently, four independent studies examined PCM spatial organization revealing the specific contribution and organization of centrosomal proteins critical for centrosome maturation in metazoans cells. Together they showed that the PCM comprises two major domains with distinct molecular composition and architecture [37–40]. Notably, these studies establish the higher-order organization of the PCM as an evolutionarily conserved property of centrosomes. PCM architecture: historical perspective, roadblocks, and recent developments Although numerous studies have revealed important structural details of the centriole and basal bodies [32–34,41], our understanding of the architecture of the PCM has remained in its infancy for over a century. The first description of the PCM dates back to the origin of centrosome biology in 1900, when T. Boveri defined it as the unstructured region where microtubules appeared to 2

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originate [112]. Decades later, convincing evidence of this initial observation was provided by Gould and Borisy, who unequivocally showed that microtubule nucleation originated from the PCM, thus defining its major functional role [42]. A central reason for this lack of progress can be attributed to the rather homogeneous electron density of the PCM on EM, which precludes detailed studies of its organization. Indeed, there is a striking paucity of evidence alluding to defined protein layers within the PCM. Most notable is the study of Paintrand and colleagues where averaging of electron micrographs exposed apparent features in the PCM region that were dependent on the centrosome purification method [43]. EM studies on saltstripped centrosomes of Drosophila and giant surf clam have also revealed an insoluble protein matrix running throughout the PCM formed from 12–15 nm elements [44,45]. This structure is resistant to treatment with high concentrations of chaotropic salts and is sufficient to allow functional reconstitution of gTuRC-mediated microtubule nucleation. Unfortunately, the molecular identity of this matrix has remained elusive and the organization of its elements is unknown. This is a common issue in EM studies, where imaging of intact centrosomes is relatively straightforward, but labeling of specific proteins by immuno-EM suffers from antigen disruption due to sectioning and antibody penetration issues. Fluorescence microscopy studies based on deconvolution microscopy have also begun challenging the notion of an amorphous PCM organization. Unlike EM, fluorescence microscopy methods readily provide molecular specificity with genetically encoded fluorescent tags and costaining with multiple antibodies, while mostly preserving antigen structure and accessibility. However, this method suffers from the very small size of the centrosome. Pioneering work from Doxsey and colleagues suggested that pericentrin is organized in a lattice structure formed from multiple interconnected rings [46] and Rattner and colleagues suggested the existence of a PCM tube around centrioles comprising the appendage proteins ninein and centriolin (CEP110) and pericentrin [47]. Yet, despite these efforts, our perception of the PCM as an amorphous, electrondense structure has populated the textbooks and has remained the dominant view until very recently. Subdiffraction-resolution fluorescence microscopy has lately emerged as a powerful tool for the investigation of the architecture of protein complexes inside cells. Three different technologies – 3DSIM, STORM and photoactivated localization microscopy (PALM), and stimulated emission depletion (STED) – have been reliably employed to study diverse cellular structures by taking advantage of different principles to circumvent Abbe’s diffraction limit [48–51]. 3DSIM relies on modulation of the excitation light by spatial patterning and, in its most practical implementation, provides a resolution gain of only a factor of two (125 nm in the x/y-plane and 250 nm in the z-plane, Box 1), yet has the least stringent requirement for fluorophore selection and readily accomodates multiple labeling [48]. Conversely, PALM and STORM methods identify fluorophore position with higher resolution (10 nm in the x/yand 25 nm in the z-plane) by Gaussian fitting methods,

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Figure 1. Subdiffraction fluorescence microscopy view of the pericentriolar material (PCM) architecture. (A) Schematic representations of the architectural elements of the interphase centrosome. The three layers of organization – centrioles and PCM fibers, and matrices – are represented separately to help visualization. The PCM is organized in two major layers of proteins: (i) molecular fibers comprising the elongated coiled-coil proteins pericentrin/pericentrin-like protein (PLP) and Cep152/Asl have their C termini near the centriole wall and their N termini extending toward the periphery; and (ii) a PCM matrix comprising Cep215/centrosomin (Cnn), g-tubulin, and Cep192/Spd2 molecules. See Figure 3 for a model with mitotic PCM. (B) Positional mapping of pericentrin domains on the interphase human centrosome. Top: Position of antibody/ probe positions on human pericentrin with predicted coiled-coil regions indicated. Bottom: Measurements of pericentrin diameter derived from 3D structured-illumination microscopy (3DSIM) micrographs of human cells stained with anti-pericentrin domain-specific antibodies. The N terminus is positioned at a distance of >500 nm from the center of the centriole. The PACT domain is position around 200 nm, a distance consistent with the centriole wall region, as measured from electron micrographs. (C) 2D projections of averages of aligned 3D volumes of human centrosomes stained with antibodies against the C- and the N-terminus regions of pericentrin. Modified from [37,38].

but necessitate more stringent control of fluorophore photoactivation and density [50,51]. Lastly, STED microscopy is based on the concept of stimulated depleted emission, which uses a powerful donut-shaped laser to bring fluorophores away from the excitation focal point thus reducing the effective point-spread function (PSF) [49]. STED reaches similar resolution to PALM/STORM methods, but requires a more sophisticated optical setup and is more challenging in 3D. All of these methods improve the resolution from two- to about ten-fold compared with diffraction-limited fluorescence microscopy thus allowing a quantum leap in the structural analysis of organelles and molecular complexes in cells, which are in large part beyond reach of classical structural biology methods (i.e., crystallography, cryo-EM) because of their size and need for cellular extraction and purification.

The analysis of the architecture of the PCM has been a particularly challenging problem because of its biochemical complexity (hundreds of components) including numerous large coiled-coil-containing proteins – most of which are completely uncharacterized structurally – and its dimensions at the boundary of the diffraction limit. Recently, several studies have started to exploit the resolving power of subdiffraction-resolution microscopy to examine centrosome/basal body molecular assembly [52,113]. The mechanism of PCM assembly: scaffolding fibers and branching matrix In novel systematic super-resolution microscopy studies, several groups demonstrated the existence of a PCM layer in the proximity of the centriole wall with a stunning, 3

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Review Box 1. Super-resolution fluorescence microscopy and 3D volume data analysis Super-resolution fluorescence-microscopy experiments generate remarkable images, but, more importantly, they can provide a substantial amount of quantitative positional information, all inside the cell. The ensemble distribution of molecules and – in many cases – domain localization and molecular orientation within individual molecules can be obtained providing an unprecedented snapshot of the organization of supramolecular complexes in cells [37,38,87,88]. Recently, powerful image-processing and analysis tools inspired by cryotomographic analyses have been implemented for the first time in super-resolution microscopy. In essence, this method aligns extracted 3D subvolumes in real space by translating, tilting, and inplane rotating the volumes to a reference by cross-correlation of voxel intensities. The process is rendered unbiased by using an average of the aligned volume as a reference for further rounds of refinement by iterative alignment. The considerable advantage of this method is that it provides excellent quantitative metrics of the spatial distribution of centrosomal components with error in the range of a few nanometers, rather than merely anecdotal examples [37]. This in turn favors precise and unambiguous protein position assignment. The subvolume image stacks can be analyzed individually, to obtain measurement variance and structural heterogeneity, as well as through volume averages. Why the latter? The derivation of an average structure enhances the structural similarities, while deemphasizing individual structural and/or staining heterogeneity. This type of analysis leads to the discovery of shared structural features and, via 3D clustering methods, could provide insights into distinct structural states of the molecular assemblies [37]. Although 3D-alignment analysis substantially improves measurement precision, the accuracy might be further improved by reducing the distance between the antigen and the fluorophore. Toward this end, an alternative to antibody primary–secondary complex (15– 20 nm) is directly labeled anti-tag nanobodies, which are considerably smaller probes (2–5 nm) and are available commercially [89]. An inherent limitation of all single-objective microscopic-imaging methods is that the derived 3D volumes have anisotropic resolution. That is, the resolution in the x- and y- planes is 2.5 times better than that in the z-plane. This introduces a distortion in the observed volumes and consequently reduces the accuracy of the distance measurements. A simple way around this is to focus on those objects oriented such that the measurements are in-plane (i.e., end-on view of centrosomes) [38–40,89]. A more precise and general approach is to correct for distortion of tilted volumes during the averaging by properly merging the object information such that rotated objects contribute axial resolution information largely missing in the other views. This is best done by a hybrid real-space/Fourier-space approach that can optimally make use of all information available from each 3D subvolume in an unbiased manner [37].

highly ordered protein architecture in human and Drosophila cells [37–40]. The PCM proximal layer – named for its vicinity to the centriole wall – is readily visible on interphase centrosomes and comprises several proteins involved in centrosome maturation (Table 1). These components display distinctive distributions: some are organized as apparent molecular fibers or toroids extending away from the centriole wall [pericentrin/pericentrin-like protein (PLP), Cep152/Asl], whereas others are organized in a matrix [Cep192/Spd-2, Cep215/centrosomin (Cnn), and g-tubulin] (Figure 1). A striking example of PCM molecular fibers is exemplified by pericentrin/PLP (Figure 1). Positional mapping using probes against different domains followed by 3D subvolume alignment and averaging revealed toroids of varying diameters [37,38] (Figure 1B,C and Box 1). Targeting the N-terminal region of pericentrin/PLP yielded larger 4

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diameters than the C-terminal regions and probes targeting the middle region produced intermediate results. This suggested that pericentrin/PLP adopts an extended conformation and that the molecules are anchored at the centriole wall region through their C-terminal PACT domain, which had been shown previously to be sufficient for centrosomal localization [53], whereas the N terminus projects outward to the periphery. Pericentrin/PLP molecules extend a remarkable distance from the centriole wall (>150 nm in human cells; Figure 1B,C) [37–40]. Importantly, STORM shows multiple PLP molecules forming clusters in the PCM region, which might follow the ninefold symmetry of the centriole [37] (Figure 2A). These findings suggest that pericentrin/PLP forms elongated fibers in the PCM and support the notion that centriole symmetry is not confined to its perimeter, but acts as an organizing principle that extends into the PCM. The coiled-coil protein Cep152/Asl has a similar orientation and distribution to pericentrin/PLP. Labeling the two termini shows that Cep152/Asl radiates outward with its C terminus at the centriole wall and the N terminus extending away [37,39]. Subdiffraction imaging shows that PLP and Asl are mostly organized in an interleaved fashion with few areas of overlap, largely excluding the formation of heteroligomeric structures through their coiled-coil regions [40] (Figure 2B). Distinct from pericentrin/PLP and Cep152/Asl, molecules of Cep192/Spd-2 are distributed rather homogenously around the centriole wall; analysis with multiple antibodies against different regions of Cep192/Spd-2 showed no clear polarity, suggesting that Cep192/Spd-2 is a globular structure organized in a tightly packed matrix. This is consistent with the lack of extensive predicted coiled-coil domains in its sequence [37–39]. Cep215/Cnn and g-tubulin molecules also appear randomly oriented around the centriole wall and organized in a matrix that can extend to the outer region occupied by the N termini of pericentrin/PLP [37–40]. Thus, in interphase centrosomes two layers of organization of the PCM are present: a proximal layer of pericentrin/PLP and Cep152/Asl, which form fibers extending up to hundreds of nanometers away from the centriole wall; and, by contrast, a matrix of interdispersed Cep215/Cnn, g-tubulin, and Cep192/Spd-2 molecules (Figure 1A). In the early phases of mitosis, during centrosome maturation, the PCM proximal layer expands into a larger outer matrix to form the fully nucleating mitotic centrosome (Figure 3). Although nearly the same set of proteins is present, their architecture in the outermost PCM layer changes. In particular, two components appear to adopt a different architecture. Pericentrin maintains its fiber-like organization around the mother centriole, but also expands into the PCM outer matrix in human cells to form the bulk of the PCM together with Cep215/Cnn [37–39]. Cep192/Spd-2 instead goes from a tight toroidal organization around the mother centriole to a matrix (Cep192)/reticular (Spd-2) structure in mitosis [37,38,40]. Notably, the PCM proximal layer is buried inside a large PCM matrix in humans and becomes apparent only after PCM fragmentation – on, for example, depletion of augmin activity – whereas it remains visible in Drosophila centrosomes, probably due to the

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Table 1. Proteins implicated in centrosome maturation Protein name TUB1G (Hu) g-Tubulin (Dm, Ce) GCP71WD, Nedd1 (Hu) Grip71WD (Dm) Cep215,CDK5RAP2 (Hu) Cnn (Dm) Spd5 (Ce) Cep192 (Hu) Spd-2 (Dm) Spd2 (Ce) Pericentrin/kendrin (Hu) PLP (Dm) Cep152 (Hu) Asterless (Dm) Plk1 (Hu) Polo kinase (Dm) Aurora-A (Hu, Dm) Air-1 (Ce) CPAP (Hu) Sas-4 (Dm) Cep97 (Hu) Cep135 (Dm) CP110 (Hu, Dm) Rcd-5/6 (Dm) Myb (Hu, Dm) Calmodulin (Hu, Dm) Map205 (Dm) PP2A (Dm)

Function in centrosome maturation Highly conserved protein required across species for the nucleation of microtubules. As part of the gTuRC, forms a template for microtubule formation at the minus ends. Involved in targeting gTuRC to the spindle and the centrosome. It copurifies in a complex with gTuRC, but also appears to have functions beyond that of a gTuRC-targeting factor. Essential protein for PCM maturation; it is the main candidate for a component of the PCM matrix that provides direct binding to most of the gTuRCs in the PCM.

Refs [11,90–93] [56,94–96] [60,97–99]

Key factor involved in both centriole duplication and PCM maturation in C. elegans, flies, and human cells. It has two defined populations, one at the centriole wall and one in the PCM.

[57,64,65,100]

Large coiled-coil protein integral to the PCM involved in centrosome maturation across metazoans. The C-terminal region contains the PACT domain, a conserved centrosometargeting region. Discovered in a mutant screen for male fertility in flies, it is primarily involved in centriole duplication by defining the site of daughter centriole formation through Plk4 kinase interaction. Asl might have a role in PCM stabilization/maintenance at the centriole. Conserved serine–threonine kinase involved in several aspects of cell division. It has been shown to phoshorylate pericentrin directly to control centrosome maturation. Serine–threonine kinase implicated in several steps of cell division, including centrosome maturation. Aurora-A is required for Cnn recruitment and phosphorylates TACC, which form a complex with Msps to stabilize microtubules at the centrosome. Conserved protein involved in centriole duplication and elongation. It might recruit PCM proteins to the centriole during maturation as part of a large, multiprotein complex. This list contains several other proteins implicated in centrosome maturation. Because their functional role and conservation remain unclear at this stage they are not be discussed in this review.

[14,62,101,102]

differences in PCM architecture compared with human cells (see below and [37,38,54,55]). 3D volume alignment and cross-correlation analysis (Box 1) of the components of the outer PCM matrix shows that they largely overlap, thus suggesting the formation of a matrix of interconnecting proteins within which specific domains of PCM components might maintain their relative spatial positioning [37,38]. What is the functional interplay of the fibers and matrices of the proximal and outer layers of the PCM during maturation? Loss-of-function experiments suggest the existence of separate but interdependent pathways for building the PCM [56,57]. A scaffolding pathway, named after pericentrin/PLP’s elongated structures, organizes the PCM proximal layer around the centriole wall and is critical for proper 3D organization of the PCM outer layer during maturation. A second pathway is mainly involved in matrix formation and relies on Cep192/Spd-2, Cep215/ Cnn, and g-tubulin, but does not appear to require pericentrin/PLP [14,37,38,40,58,59,114]. In interphase centrioles, pericentrin/PLP appears to be at the top of the hierarchy of the PCM assembly of the proximal layer. Pericentrin/PLP is required to recruit Cep215/Cnn [14,37,38,59,114], but likely not vice versa [38,60,61]. In turn, pericentrin is sufficient to recruit gTuRCs in the absence of Cep215 [38]. These activities might be determined by pericentrin/PLP directly binding to Cep215/Cnn and g-tubulin [46,62,63]. During centrosome maturation, pericentrin and Cep215/Cnn are the first to expand into a large matrix in G2, but this is not sufficient for full centrosome maturation, which requires Plk1

[67,82–84,103,104]

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phosphorylation of pericentrin. Interestingly, only a subpopulation of mitotic pericentrin located close to the centriole is phosphorylated and is required for recruitment of Cep192 and g-tubulin [58]. In addition, overexpression of pericentrin during interphase is sufficient to recruit Cep215 and build a massive PCM matrix similar to mitotic centrosomes. These observations support the notion that the pericentrin layer proximal to the centriole wall has an important role in PCM expansion under regulatory control. Conversely, in Drosophila, the pericentrin homolog PLP maintains its fibrous/toroid structure around the mother centriole, but does not expand into the outer PCM matrix during centrosome maturation. This would suggest that pericentrin might have acquired during evolution a polymerizing molecular activity much like Cep215/Cnn that Drosophila PLP lacks and explains the less dramatic effect of PLP depletion compared with pericentrin in centrosome maturation in flies [14,37,38,59,114]. Notably, a population of Cep215/Cnn, Cep192/Spd-2 and g-tubulin is recruited to mitotic and interphase centrosomes in the absence of pericentrin/PLP [37,38,56, 57,59,61], indicating that Cep215/Cnn can self-assemble and/or have other molecular interactions independently from pericentrin (i.e., with Cep192/Spd-2) that are sufficient to form a reticular structure. This suggests the existence of a second pathway that appears to be mainly involved in matrix formation and relies on Cep192/Spd2, Cep215/Cnn, and g-tubulin. Indeed, RNAi and genetic mutants show that Cep192/Spd-2 molecules are critical for Cep215/Cnn matrix formation, thus making Cep192/Spd-2 5

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Figure 2. Nanometer-scale organization of the pericentriolar material (PCM) proximal layer. (A) Stochastic optical-reconstruction microscopy (STORM) images of S2 cells stained with antibodies against pericentrin-like protein (PLP), the Drosophila pericentrin ortholog. Left: End-on view. Right: Side view. Note the quasi-nine-fold symmetric distribution of PLP. (B) 2D projections of 3D structured-illumination microscopy (3DSIM) images of Drosophila S2 cells stained with antibodies against Plk4 and PLP. Note the interleaved distribution of PLP and Plk4 on interphase centrioles. The N termini of Asl have been shown to have a similar distribution to Plk4 on interphase S2 cells and the two proteins interact directly in vitro. (C) Overview of the experimental scheme used for 3D volume alignment and averaging of subdiffraction-resolution images. See Box 1. Modified from [37].

the main candidate to drive Cep215/Cnn oligomerization [57,64–66]. Surprisingly, in flies Spd-2 does not reach the further region of the PCM where Cnn forms a matrix, but it does have a reticular structure that might play a branching role in a subregion of the PCM [37,40]. Alternatively, Cep192/Spd-2 might promote PCM matrix formation through recruitment of a regulatory factor such as Plk1 [40,66]. The existence of two separate pathways is further supported by studies in humans and flies that show that Cep192/Spd-2 is important for Cep152/Asl recruitment [65,86], but not Plp [65]. In addition Plp and Asl are not required for the localization of each other [37,57,65,82]. Altogether, these experiments suggest that pericentrin/ PLP and Cep192/Spd-2 are indirectly linked in the PCM and provide more evidence for the notion that Cep192/Spd2 and pericentrin/PLP lie in independent pathways for PCM organization through their mutual ability to interact with Cep215/Cnn. The experimental evidence summarized above is consistent with a combination of a template and self-organization model for PCM formation [68]. The proximal interphase PCM layer, which is formed from pericentrin/ PLP fibers around the mother centriole, provides a molecular scaffold that recruits Cep215/Cnn. In turn, this scaffold provides a template for the mitotic outer matrix expansion. This process is driven by more accumulation of Cep215/Cnn, Cep192/Spd-2, and, in human cells, pericentrin. 6

The dynamics of PCM assembly and the role of kinases How is PCM recruitment regulated in space and time during centrosome maturation? As mentioned above, in human cells robust centrosome maturation requires Plk1dependent phosphorylation of a subset of pericentrin molecules in the vicinity of the centriole wall and the subsequent recruitment of Cep192 and g-tubulin [58]. Consistently, Plk1/Polo is also localized in proximity to the centriole wall [40,58]. Interestingly, Plk1/Polo is also required for Cnn phosphorylation, suggestive of a direct role for these kinases in Cnn regulation during PCM expansion [40,56,58,59]. Fluorescence recovery after photobleaching (FRAP) experiments in fly embryos show that Cnn molecules are dynamically associated with the centrosome and are first recruited near the centriole, before spreading to the centrosome periphery [69]. By contrast, FRAP studies in fly embryos indicate that the centriole-associated PACT domain of centriolar PLP does not exchange rapidly [14]. Because the pericentrin/PLP population at the centriole wall is important for maturation, it would be interesting to examine whether PLP can also be phosphorylated by Polo (like its mammalian counterpart), if so possibly unveiling a conserved regulation mechanism in PCM recruitment, in which phosphorylated pericentrin/PLP at the centriole wall provides structural scaffolding for PCM expansion. Interestingly, PLP loss of function has opposite effects on PCM recruitment on the daughter (apical) compared to the mother (basal) interphase centrosomes in Drosophila neuroblasts suggesting a

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Figure 3. Pericentriolar material (PCM) architecture during centrosome maturation. Schematic representation of a mammalian centrosome during centrosome maturation. Note the expansion of the PCM proximal layer of interphase cells during G2/M through the formation of an outer matrix of Cep215, pericentrin, and Cep192 molecules. g-Tubulin ring complexes (gTuRCs) are embedded within the PCM and promote microtubule nucleation during mitosis. By contrast, PCM expansion in Drosophila does not require the formation of a matrix comprised of the pericentrin homolog PLP, but only of its centriole associated component.

special form of regulation relying on Polo localization in stem cells whose mechanism remains unclear [112]. The serine–threonine kinase Aurora-A is also implicated in centrosome maturation [70–72]. Although its activity is required for mitotic entry through regulation of Cdk1 and Plk1 kinases in a feedback loop through the protein Bora [73–75], its function in centrosome maturation appears distinct from Plk-1. Aurora-A and Cnn are mutually dependent for their centrosomal recruitment during maturation and this activity is mediated through a direct interaction between Aurora-A and a Cnn C-terminal region [76]. Aurora-A also stabilizes microtubule growth by phosphorylation of TACC, a protein in complex at the centrosome with the microtubule regulator MSPS [77,78]. It will be of interest to better understand the interplay between these kinases and the full set of substrates they regulate during centrosome maturation. Recent work suggests that some PCM components implicated in centrosome maturation (PLP, Asl, Cnn) are part of a large protein complex containing tubulin and Sas-4, which acts as an attachment factor to the centriole wall [79,80]. Although these complexes were shown to associate in embryonic extracts, it remains unclear whether they are assembled in the cytoplasm before centrosomal loading or whether they derive from PCM disruption during purification. Although PLP, Asl, and Cnn are present together on mother centrioles in Drosophila interphase cells, these

PCM proteins are recruited to daughters at distinct times during mitosis, with Sas-4 recruited first to the daughter centriole and subsequently, but not simultaneously, Asl and PLP around metaphase, followed by the outer matrix components Cnn and g-tubulin [37]. It is possible that Drosophila cells and embryos have different timing mechanisms for recruitment of these proteins, owing to the different time scales of cell division. Alternatively, it is possible that only a small subpopulation of these proteins might be forming a bona fide complex while transported together to the centrosome or when all are located at the centrosome after docking [16,81]. Platforms and gates in centriole duplication Studies from worms to humans suggest that mother centrioles act as an assembly ‘platform’ for the formation of daughter centrioles during their duplication. This process comprises the sequential recruitment of Cep192/Spd-2, Cep152/Asl, Plk4, Sas-6, and CPAP/Sas-4, which allows procentriole assembly [34,82–85]. Subdiffraction-resolution analysis together with 3D subvolume alignment and averaging of centrosomes in G1 and G2 shows that Cep152/ Asl and pericentrin/PLP undergo a pronounced change in architecture by forming a zone of exclusion on mother centrioles, which correlates with the position where the daughter emerges [37,38]. These observations suggest a multistep mechanism that involves changes in PCM molecular fiber organization. This includes the selection of the site on the mother centriole where daughter centrioles will emerge on the recruitment of Cep152/Asl structures by Plk4 and subsequent disassembly of a subpopulation of Cep152/Asl and pericentrin/PLP, to allow growth of the daughter centriole [37–39,86]. This molecular gap, which is present in human and flies, is observed in Asl and PLP structures when labeled using anti-gfp nanobodies (2 nm in size), thus probably excluding the possibility of lack of antigen accessibility to antibody binding (unpublished observations). Concluding remarks Our understanding of PCM architecture has changed substantially through the recent advancements of subdiffraction imaging. Although the existence of an organized layer of proteins around centrioles (PCM tube) [4,15,47] had been previously hypothesized, the findings highlighted in this review reveal the existence of multiple layers in the PCM. More precisely, they expanded the view of pericentrin architecture – previously described as a lattice-like structure [46] – and function by revealing the dual nature of pericentrin distribution (mitotic matrix-like versus interphase fibers) as well as the orientation of its molecules within the PCM. In addition, they provide a systematic analysis – with a quantitative method with nanometer precision – to establish the position and orientation of a large number of proteins critical for various facets of centrosome biogenesis. These studies further explain the structural heterogeneity of the components closely associated with the centriole wall during the cell cycle by showing that their state (open versus closed) is correlated with daughter centriole formation. Lastly, and critically, they provide a conceptual framework that will greatly facilitate 7

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Box 2. Outstanding questions  Several fundamentally important but unanswered questions remain. Is Cep215/Cnn capable of self-assembly? What are the precise molecular roles of the other PCM components like pericentrin/PLP and Cep192/Spd-2 in building the PCM matrix around interphase centrioles, how are these relationships modulated during centrosome maturation, and how are they regulated by the cell cycle machinery? Are Cep192/Spd-2 and pericentrin/PLP required to alter the connectivity, the orientation, and/or the kinetics of Cep215/Cnn assembly? To answer these questions it will be important to determine whether the set of molecular interactions underpinning PCM organization during interphase differs from that in mitosis. Although pericentrin/PLP and Cep215/Cnn interactions appear to be maintained throughout the cell cycle, evidence suggests that Spd-2 might play a major role in PCM assembly during mitosis but not interphase [38]. Recent work suggests that Cep192 participates in the phospho-regulation of NEDD1 and controls its ability to associate with and recruit g-tubulin to mitotic centrosomes, raising the tantalizing possibility that Cep192 contributes to PCM activation at the onset of mitosis [111]. A systematic biochemical analysis of the interactions with recombinant material or subcomplexes purified from cell extracts at various stages of centrosome assembly will be necessary to answer these questions directly. Considering the importance of microtubule organization and anchoring in specialized cellular processes, including stem-cell division, it would be important to analyze the similarities and differences in PCM organization during interphase in cells about to undergo symmetric versus asymmetric divisions [5,114].  During metazoan evolution, pericentrin/PLP has acquired the ability to be recruited not only at the centriole wall as a fiber (in human and Drosophila cells) but also as a matrix component in the outer PCM during maturation (in human cells). This more recently acquired property of pericentrin is under regulatory control. Indeed, overexpression of pericentrin in human cells can lead to matrix formation in interphase centrosomes, which in normal conditions have only a fiber/toroid distribution. Interestingly, this function is not required to make the outer matrix in Drosophila – or in Caenorhabditis elegans, which seems to lack a pericentrin homolog altogether. This would suggest that the original primary role of pericentrin/PLP is to provide an interphase scaffold for PCM recruitment and that later in evolution it gained additional functionality to facilitate PCM expansion in the outer matrix.

understanding of centrosome maturation at the structural level. The future challenge is to integrate the compositional, functional, and structural data into a coherent view of PCM assembly – see Box 2 for analysis of outstanding questions. A major task is to bridge the nanometer-resolution information obtained from super-resolution microsco˚ ngstrom resolution maps derived from py with the A classical structural methods. Integral to this task is the generation of probes targeting various regions of all centrosomal proteins and the isolation or reconstitution of protein complexes from purified components or extracts. In addition, the molecular role of individual components in the kinetics of PCM assembly will be clearly defined via in vitro reconstitution, enabling the basic principles of PCM formation to be elucidated. These studies should be guided by parallel efforts in cells aimed at defining a high-resolution map of the dynamics of PCM proteins and a comprehensive analysis of their regulatory networks. In conclusion, recent studies highlighted here demonstrate the power of combining multimodal subdiffractionresolution microscopy with quantitative image analysis to reveal molecular-scale details of organelle architecture. Importantly, they defined a fundamental map of PCM 8

 Pericentrin/PLP is expressed in metazoans in many isoforms and splicing variants [14,102]. Super-resolution imaging seems unable to resolve the individual isoforms, suggesting that the different pericentrin/PLP species are arranged in close proximity or bundle together into a multimolecular fiber at the centrosome with a symmetry likely dictated by the centriole nine-fold structure (see Figure 2D in main text) [37]. In vitro biochemical and structural analysis will be critical to demonstrate the existence of a pericentrin/PLP multimeric structure and to define the biochemical determinants of its formation and the role of the different isoforms in higher-order PCM matrix organization.  Cep152/Asl on interphase centrosomes is located in the same PCM domain as pericentrin/PLP and has a similar elongated orientation, but neither pericentrin/PLP nor Cep152/Asl is required for the recruitment of the other, implying independent anchoring to the centriole wall [37,86]. Do the Cep152/Asl elongated structures have a role in organizing the PCM or only in providing a platform for duplication? Mutant analysis of Drosophila spermatocytes suggests that Asl is indeed necessary for PCM stabilization [67,104], whereas RNAi studies suggest that it is not [40]. One possibility is that Asl has an indirect role in PCM organization by recruiting Spd-2 at the centriole wall, which might affect Spd-2 matrix organization. Further studies are required to address this issue.  How do pericentrin/PLP and Cep152/Asl fibers bind to the centriole wall? Although Asl has been shown to bind Sas-4 directly [82], it remains unclear how PLP is anchored to the centriole wall. Because pericentrin/PLP appears to be distributed symmetrically around the centriole, it is likely to take advantage of the microtubule doublets/ triplets, directly or through an attachment factor, or of the space between [37,38]. Interestingly, pull-down interaction experiments suggested that Sas-4 N termini might link PLP to the centriole wall [80].  Recent discoveries defined Cep215 as the major interaction partner and regulator of gTuRC activity in the PCM [60,99]. In addition, the N-terminal domain (NTD) of pericentrin/PLP has been shown to bind gTuRC directly, albeit weakly, through GCP2/3 [46,63], where it may help activate gTuRCs for microtubule nucleation. This putative globular domain does not seem to be necessary for PCM maturation and its precise role remains unclear [37]. Further studies will be required to assess whether pericentrin and gTuRC are indeed part of a bona fide complex and, if so, what their functional role is in centrosome maturation and PCM organization.

organization and revealed a functional framework to dissect how its organization is achieved. In-depth understanding of the centrosome and basal body architecture and function is crucial for the discovery and validation of protein targets as determinants of disease. Acknowledgments The authors sincerely apologize to authors whose studies they could not cite due to space constraints.

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